Coronary microvascular disease (MVD) is prevalent in as many as 3 million American women with ischemic symptoms without obstructive coronary artery disease (CAD). Despite advances in recent conventional diagnoses and cardiovascular angiography, the clinical diagnosing and treating of coronary MVD remains disappointing. The mechanism of coronary MVD is still not well understood, which lead to missed or misdiagnosis. Different from traditional epicardial CAD, diffuse endothelial dysfunction resulting from various reactive oxygen species (ROS) is a pathological hallmark of MVD, which in turn triggers focal pro-thrombosis and anti-fibrinolysis. Activated FXIII (FXIIIa) plays a key role in the stabilizatio of thrombosis by mediating fibrin-fibrin and fibrin- 2-antiplasmin cross- links. Recently, fibrin has been detected as a major composite in acute myocardial infarction and increases with ischemic time. Here, we believe that MVD-induced ischemia results from microthrombi and FXIII may play a key role during this process. We hypothesize that FXIII is a specific biomarker for coronary MVD and its activity can be evaluated ex vivo and in vivo by a FXIII targeted radiolabeled probe. At a mechanistic level, we also hypothesize that the microvasculature might be more sensitive to oxidative stress -induced endothelial dysfunction than macro-vasculature. Evaluation of increased FXIII activity may predict long-term functional outcomes, such as decreased wall motion and impaired cardiac function. We will test these hypotheses with a combination of ex vivo and in vivo methods, including; 1) cellular and molecular studies in primary ECs derived from micro- and macro-vasculature, 2) application of a novel model of PDT-induce MVD in mice, 3) application of a non-invasive molecular imaging method to monitor the dynamic activation of FXIIIa in the setting of MVD, and 4) application of a state-of-the-art microCT method for the assessment of cardiac function and wall motion as long-term end-points for the evaluation of MVD. As such, the proposal is expected to establish a clinically relevant animal model of coronary MVD and validate a molecular imaging probe for the detection and risk stratification of coronary MVD. The information generated from these studies may substantially alter our current practice for the evaluation and management of coronary MVD. PUBLIC HEALTH SIGNIFICANCE: Cardiovascular disease is the leading cause of death. Endothelial dysfunction often triggers thrombotic cascade and inhibits thrombolysis to facilitate blood clotting, causing multiple ischemic lesions in the heart; hence, its study is extremely important to clinicians. The results of this project will provide direct evidence to monitor in viv coronary clots non-invasively. Thus, the FXIIIa within clots will serve as a diagnostic biomarker to determine biological aging to guide treatment, to evaluate the efficacy of fibrinolysis, and to predict the prognosis of pathology.